Biologically active compounds from selected aphyllophorales mycelial cultures

DOI: 10.2478/fobio-2014-0004

Acta Universitatis Lodziensis

Introduction

Representatives of Basidiomycota are

an important source of active compounds of the multidirectional therapeutic activity; inter alia, antiviral, bacterio- and

Biologically active compounds from selected aphyllophorales

mycelial cultures

KATARZYNA SUŁKOWSKA-ZIAJA*,BOŻENA MUSZYŃSKA &ANNA FIRLEJ

Department of Pharmaceutical Botany, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Kraków, Poland

E-mail: katarzyna.sulkowska-ziaja@uj.edu.pl

ABSTRACT

For a long time fungi belonging to Basidiomycota phylum have been in the center of attention because of the presence in their fruiting bodies of compounds with known therapeutic activity. Mycelial cultures of two aphyllophorales species occurring in Poland, Hydnum repandum L., and Sparassis crispa (Wulf.) Fr., were analyzed in our study. The main aim of the study was qualitative and quantitative analysis of extracts obtained from the mycelial cultures for the presence of known biologically active compounds, including phenolic acids, non-hallucinogenic indole compounds and sterols.

For analyses a reversed-phase chromatography (RP-HPLC) method was used. The presence of eight phenolic acids including gallic, gentisic, p-hydroxybenzoic, caffeic, p-coumaric protocatechuic, syringic, vanillic and cinnamic acids was confirmed in the extracts obtained from the biomass. The quantitatively predominant metabolites in biomass from in vitro cultures of H. repandum and S. crispa were protocatechuic acid (6.23 g/g DW) and p-hydroxybenzoic acid (4.52 g/g DW). Derivatives of indole such as indole, serotonin, tryptamine and tryptophan were measured quantitatively. Their total content was estimated as 1.28 g/g DW and 3.07 g/g DW in H. repandum and S. crispa extracts, respectively. The major metabolite found was tryptophan. In addition, ergosterol, one of the sterols present in the biomass of in vitro cultures of S. crispa was analyzed (700.87 g/g DW).

The obtained results confirm the hypothesis that mycelial cultures of domestic species of aphyllophorales are able to accumulate biologically active metabolites.

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fungistatic, antiparasitic, as well as antitumor, anti-inflammatory, vessel protective or hypoglycemic (Zjawiony 2004, Sułkowska-Ziaja et al. 2005). More and more research has been undertaken to identify the chemical composition of not only fruiting bodies, but also the mycelium extracted from in vitro cultures. Studies in this area have demonstrated the existence of qualitative and quantitative differences in the production of certain groups of chemical compounds between fruiting bodies and mycelium from in vitro cultures. This is important for both cognitive and application reasons.

The present study involved two wild edible mushrooms commonly growing in Polish forests: Hydnum repandum L.

(Hydnaceae) and Sparassis crispa (Wulf.) Fr. (Sparassidaceae). These species belong to an artificial systematic group known as Aphyllophorales.

The aim of the study was to determine the levels of biologically-active compounds in extracts from the biomass of in vitro cultures. It is a continuation of an extensive analysis of the chemical composition of biomass from mycelial cultures to confirm their usefulness as potential sources of compounds with biological properties. As far as we know, our study is the first one where the above-mentioned groups of compounds in the biomass from in vitro cultures of the examined fungi species have been described.

Material and methods Fungal material

Samples of mushrooms were collected in 2012 in mixed forests of northern Poland. Taxonomic identification was conducted according to Gumińska and Wojewoda (1988). Representative voucher samples were deposited in the Department of Pharmaceutical Botany UJCM, Kraków, Poland. Mycelial cultures were derived from explants originating from the hymenial part of fruiting bodies. Fruiting

body fragments were sterilized, placed on Petri dishes with modified Oddoux medium (Oddoux 1957), incubated at a temperature of ±22°C and subcultured every three weeks. Experimental in vitro cultures were maintained in Erlenmayer flasks, containing 250 mL of medium and shaken at a rate of 140 rpm. The cultures were maintained for three weeks. Then, the biomass was separated from the medium, frozen and lyophilized. Determination of phenolic acids

The amount of 5 g of powdered material was hydrolysed with 2 M hydrochloric acid for 2 h at 100° C. Hydrolysates were extracted with 50 mL of ethyl acetate and concentrated to dryness. HPLC analyses were conducted using an HPLC VWR Hitachi-Merck apparatus: L-2200 autosampler, L-2130 pump, LiChrospher RP-18e column (250mm×4mm, 5µm) at 25° C, L-2350 column oven, and L-2455 diode array

detector at UV range 200-400nm. The mobile phase consisted of solvent A: methanol/0.5% acetic acid 1:4 (v/v), and solvent B: methanol. The gradient was as follows: 100:0 for 0–25 min; 70:30 for 35 min; 50:50 for 45 min; 0:100 for 50-55 min; 100:0 for 57–67 min. (Ellnain-Wojtaszek & Zgórka 1999). Phenolic acid standards were purchased from Fluka (Chemie AG) and Sigma (St. Louis, USA).

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Another 5 g of powdered material was extracted with 100 mL of methanol for 2 hand mixed extracts, which were then concentrated to dryness. The HPLC study was performed according to the procedure described by Muszyńska (Muszyńska et al. 2009). Briefly, the conditions were as follows: Hitachi

HPLC apparatus; L-7100 pump; Purospher RP-18 column (250 mm × 4 mm, 5 µm); solvent system – methanol : water : ammonium acetate 15:14:1 (v/v/v); flow rate - 1ml/min; and UV detector ( = 280nm). Indole standards were purchased from Sigma (St. Louis, USA).

Determination of sterols

The third 5 g portion of powdered material was mixed with 100 mL of a 75:25 (v/v) mixture of methanol and dichloromethane, followed by sonification for 10 min and centrifugation at 10 000 rpm for 5 min. Combined extracts were concentrated to dryness. The HPLC method was performed according to the procedure developed by Yuan (Yuan et al. 2008). The mobile phase consisted of solvent A:

methanol : water 20:80 (v/v), and solvent B: methanol : dichloromethane 75:25 (v/v). A gradient procedure was used as follows: starting at sample injection, 60% of B for 5 min; a linear gradient from 60 to 100% of B for 10 min; and 100% of B for 10 min. The flow rate was 1.0 mL/min. Sterol standards, ergosterol, and ergocalciferol were purchased from Fluka (Chemie AG).

Results

The results of the analyses are shown in Table 1. Amongst the fifteen studied phenolic acids, five were detected in the extracts from the biomass from an in vitro culture of H. repandum; additionally, cinnamic acid was identified. In turn, seven phenolic acids were found in similar extracts of S. crispa. The total amounts of phenolic acids and cinnamic acid were 14.44 g/g and 12.65 g/g DW in biomass from H. repandum and S. crispa, respectively (Tab. 1). A sample chromatogram shows the separation of phenolic acids in the extract from of H. repandum biomass (Fig. 1).

Among the 12 indole compounds analyzed, three were detected in the extracts from H. repandum as well as S. crispa; i.e. indole, tryptamine and L-tryptophan. In addition, trace amounts of serotonin were detected in both extracts. The total amounts of indole compounds were similar (0.98 g/g and 1.19 g/g DW in H. repandum and S. crispa extracts, respectively) (Tab. 1). Ergosterol was the only representative of the analysed compounds with a sterol structure detected at level 700.87 g/g DW, in S. crispa extracts.

Discussion

Earlier studies of the chemical composition of the fruiting bodies of the investigated species showed very similar

qualitative compositions for all investigated metabolites; however, in the biomass of the in vitro cultures, the level

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of determined compounds was slightly lower (Sułkowska-Ziaja et al. 2014). Phenolic acids determined in the current studies are characterized by a wide spectrum of biological activity. The strongest antioxidant activity is exhibited by vanillic acid and in a cinnamic acid derivative – caffeic acid. Gallic, p- hydroxybenzoic and protocatechuic acids detected in H. repandum and also S. crispa biomass are characterized by documented, multidirectional activity:

antioxidant, antibacterial, antiviral, anti-inflammatory or antifungal. Furthermore, the protocatechuic acid found in the largest amounts in the conducted studies is characterized by immunomodulatory, spasmolytic, cardioprotective and antithrombotic activity (Ferreira et al. 2009). A previously conducted chemical analysis of fruiting bodies also showed the quantitative predominance of this metabolite.

Table 1. Contents of biologically active compounds in mycelial cultures (g/g DW).

Metabolites Hydnum repandum Sparassis crispa

Indole compounds Indole 0.018±0.05 0.07±0.02 Serotonin * * Tryptamine L-Tryptophan 0.02±1.05 0.94±0.09 0.05±0.11 1.07±0.17 Phenolic acids Gallic acid 4.16±0.12 2.27±0.34 Gentisic acid 2.03±0.11 0.10±0.01 p-Hydroxybenzoic acid nd 4.52±0.4 Caffeic acid nd 0.10±0.16 o-Coumaric acid nd 1.45±0.21 Protocatechuic acid 6.23±0.02 3.92±0.3 Syringic acid 0.41±0.21 0.29±0.02 Vanillic acid 0.60±0.56 nd Cinnamic acid 1.01±0.01 nd Sterols Ergosterol nd 700.87±0.3 Ergocalciferol nd nd

Values are means of three experiments ± SD, nd - not detected, *- traces.

Indole-derived non-hallucinogenic compounds are also an interesting group of secondary metabolites found in fungi. Estimating the content of this group of metabolites in the investigated species seems to be important due to their biological activity as well as from a toxicological viewpoint (Muszyńska et

al. 2011a, b). Within the scope of the study, the determined compounds have important biological functions as natural neurotransmitters and neuromodulators (serotonin, tryptamine), and are involved in the regulation of circadian rhythms, body temperature, mood, blood pressure, and appetite (5-hydroxy-tryptophan,

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(serotonin, melatonin). L-tryptophan and 5-hydroxytryptophan exhibit a hypnotic, antidepressant activity. In turn, melatonin and hydroxyindole derivatives have antioxidant potential.

The data concerning the appearance of non-hallucinogenic indole compounds in the representatives of Basidiomycota mainly refer to tryptophan, a biogenetic progenitor of all other indole compounds. Tryptophan is one of the diet supplements used as antidepressants. It can cross the brain-blood barrier and in the central nervous system can be converted to serotonin and melatonin, important neurotransmitters and neuromodulators controlling the circardian rhythm. A rich source of these

metabolites are edible mushrooms e.g. Suillus bovinus where tryptophan content is 25.9 mg/100g d.w.

The last study demonstrates the occurence of a biogenic amine, tryptamine in many basidiomycetes. Tryptamine, synthesized from tryptophan is a direct precursor of different active compounds, including serotonin. Considerable quantities of tryptamine were detected in Suillus luteus (34/100d d.w.) and Leccinum rufum (31.71 mg/100g d.w.) (Muszyńska et al. 2011a, b). It has multi directional pharmacological activity i.a. it takes part in body temperature regulation, moodiness, organism maturation but also tissue regeneration and cell senescence.

Figure 1. HPLC chromatogram of phenolic acids in biomass of mycelial culture of Hydnum repandum (1-gallic acid, 2-protocatechuic acid, 3-gentisic acid, 4-vanillic acid, 5-syryngic acid, 6-cinnamic acid).

One of the main components of fungal cell membranes – ergosterol – is converted into vitamin D in the presence of sunlight or another ultraviolet light source (Mattila et al. 2002). This vitamin plays an essential role in the prevention of cancer, through increasing tumor cells

phagocytosis and facilitating other immunomodulatory functions. According to the latest studies, vitamin D also blocks the angiogenesis in tumors and impairs their growth progress (Kopczyński 2012, Mraz et al. 2010). The highest levels of ergosterol have

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been noted in saprotrophic fungi and may constitute up to 83–89% of the entire amount of sterols. In addition, it has promising anti-allergic and immunostimulatory properties. Therefore, the search for new sources of this compound is becoming more and more popular (Yuan et al. 2006). Our research has shown that S. crispa is one

of the fungi with significant ergosterol levels in mycelial cultures. The data presented in this paper confirm the significant potential of chemical components with recognized antioxidant activity. The species can be considered an alternative source of phenolic acids and ergosterol.

References

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Streszczenie

Grzyby z gromady Basidiomycota od dawna budzą zainteresowanie ze względu na występowanie w ich owocnikach szeregu związków o uznanych właściwościach leczniczych. Obiektem przeprowadzonych badań były kultury mycelialne dwóch gatunków grzybów afylloforoidalnych występujących na terenie Polski Hydnum repandum L. oraz Sparassis crispa (Wulf.) Fr. Celem podjętych badań była jakościowa i ilościowa analiza ekstraktów otrzymanych z kultur mycelialnych pod kątem

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Do oznaczeń wykorzystano wysokosprawną chromatografię cieczową faz odwróconych (RP-HPLC). Na podstawie analizy stwierdzono w ekstraktach z otrzymanej biomasy obecność ośmiu kwasów fenolowych: galusowego, gentyzynowego, p-hydroksybenzoesowego, kawowego, kumarowego, protokatechowego, syryngowego, wanilinowego oraz kwasu cynamonowego. Ilościowo dominującym związkiem był kwas protokatechowy w ilości 6,23 g/g s.m. (H. repandum) oraz kwas hydroksybenzoesowy w ilości 4,52 g/g s.m. (S. crispa). Spośród związków pochodnych indolu ilościowo oznaczono: indol, serotoninę, tryptaminę i tryptofan. Całkowita ich zawartość wynosiła 1,28 µg/g s. m. (ekstrakty z H. repandum) oraz 3,07 µg/g s.m. (ekstrakty z S. crispa). Ilościowo dominującym metabolitem był tryptofan. Spośród steroli oznaczono ergosterol w biomasie z kultur in vitro S. crispa (700,87 µg/g s.m).

Uzyskane wyniki wskazują, że przebadane kultury mycelialne krajowych gatunków grzybów afylloforoidalnych są zdolne do akumulacji metabolitów aktywnych biologicznie.